The present invention provides unique methods and apparatus for identifying microscopic particles, such as protozoa and other microbes suspended in a fluid or gas.
Currently accepted methods for identification of pathogenic microscopic particles require relatively long, labor-intensive process. For instance, to determine whether Cryptosporidium parvum or Giardia lamblia is present in drinking water, suppliers must employ the USEPA method 1622, a long and labor-intensive procedure. Clinical laboratories and food inspectors also must use long labor-intensive procedures to locate and identify harmful bacteria.
Unfortunately, there are many circumstances when positive identification of a microbe cannot wait. A contamination of drinking water by Cryptosporidium must be recognized immediately, before the water is delivered to homes. Likewise, identification of a specific cause of a disease, such as bacterial meningitis, many times cannot wait the hours required. Finally, detection and identification of bacteria in food sources, such as beef, takes so long that in most cases, the food is distributed before the problem is discovered.
A variety of methods and apparatus exist for detection of microscopic organisms. For instance, De Leon, et al. in U.S. Pat. No. 5,770,368 teaches Cryptosporidium detection methods. The viability or infectivity of the encysted forms can be determined by synthesizing a cDNA from an induced HSP RNA template using a primer that is specific for particular genus or species of protozoa, followed by enzymatic amplification of cDNA. Alternatively, infectivity can be determined by amplifying HSP DNA from infected cells using a primer pair that is specific for a particular genus or species of protozoa.
Steele, et al. in U.S. Pat. No. 5,693,472 discloses detection of Cryptosporidium parvum. A method and kit for the detection of Cryptosporidium parvum in aquatic and biological samples such as surface water or feces is described. The method relies on the use of primers to detect all or a portion of at least one DNA sequence characteristic of Cryptosporidium parvum, the sequence being all or part of the genomic regions referred to as 38G and HemA contained within recombinant plasmids pINV38G, and pHem4, respectively.
Pleass, et al. in U.S. Pat. No. 5,229,849 discloses laser Doppler spectrometer for the statistical study of the behavior of microscopic organisms. An improved method and system of monitoring and identifying microbiota swimming in a fluid or moving across surfaces in a fluid provides a sensitive method for rapidly measuring very small changes in activity, and detecting and identifying individual microbes in relatively large volumes of fluid, even in the presence of detritus. The system comprises a laser station, a sample collector station, a picture taking station and a monitoring station.
Wyatt, et al. in U.S. Pat. No. 4,548,500 teaches process and apparatus for identifying or characterizing small particles. An apparatus and process are described for the characterization and/or identification of individual microparticles based upon the measurement of certain optical observables produced as each particle passes through a beam of light, or other electromagnetic radiation. A fine beam of, preferably, monochromatic, linearly polarized light passes through a spherical array of detectors, or fiber optics means, to transmit incident light to a set of detector means, and a stream of particles intersects the beam at the center of the spherical array. Selected observables calculated from the detected scattered radiation are then used to recall specific maps, from a computer memory means, one for each observable.
Lee, et al. in U.S. Pat. No. 5,473,428 disclose an interferometric temperature sensing system having a coupled laser diode wherein the magnitude is adjusted corresponding to a prior feedback laser beam. An interferometric temperature sensing system provides a simplified design for accurately processing an interference fringe pattern using self coupling effects of a laser detection element, where a laser diode and an optical detection element are combined in one package.
Curtis Thompson""s U.S. Pat. No. 5,582,985 teaches detection of mycobacteria. The invention provides a method, compositions, and kits useful for detecting mycobacteria in a sample. The method includes contacting the sample with a formaldehyde solution, an organic solvent, and a protein-degrading agent prior to hybridizing a mycobacteria-specific nucleic acid probe to the sample. The invention has particular utility in detection and susceptibility screening of human-disease causing mycobacteria such as mycobacterium tuberculosis.
The unique system of the present invention provides accurate and valid measurements for identifying a wide variety of microscopic particles, such as protozoa and other microbes suspended in a fluid or gas. The inventive methodology provides a procedure for the quantitative and qualitative identification of particle species derived from measurement of light scattered by the particle that is collected by an array of optical sensors surrounding the suspended particle, in a convenient and reliable manner.
In more detail, the light scattered by the suspended particle is detected by the sensor array and converted to an electrical signal, e.g. a voltage. The voltage from each sensor is entered into a modifying means component where the voltages are digitized and the resulting values are used as fingerprints for particle identification. The unique modifying component comprises prediction formulas derived from one or more sets of empirically determined one-dimensional or multi-dimensional probability histograms that are functions of one or more mathematical combinations of the digitized voltages. Each set consists of individual probability histograms, which give the likelihood that observed values of specific combinations of digitized voltages were produced by a specific particle species. Thus, the unique modifying component of the inventive system interprets the measured signals as xe2x80x9cspecies specificxe2x80x9d when the prediction formulas result in probability values that are large for a specific species.
In one embodied form, the inventive method for rapidly detecting and identifying microscopic particles for quantitative and qualitative measurement comprises the steps of:
a) suspending the particle to be identified in a control fluid contained within a sample chamber;
b) holding the sample chamber in a prescribed orientation with respect to an intense light source;
c) illuminating the sample chamber with said light source;
d) collecting and measuring the scattered light from the sample chamber by means of an array of optical sensors surrounding the sample chamber;
e) converting a voltage output from the array of sensors to a digital signal as the particle passes through the intense light source; and
f) comparing the derived signal with a library of probability histograms and statistically classifying the resultant data to identify the microscopic particles present.
In accordance with the present invention, the library consists of histograms for each particle species encompassed by a statistical classification algorithm that calculates the probabilities that the associated signal was produced by those particle species. The probability histogram is derived empirically from a measure of the frequency that a species of microparticle is associated with a specific range of values of a mathematical combination of the digitized sensor voltages. Thus, the frequency-of-occurrence histogram can be produced for one mathematical combination, i.e., a one-dimensional analysis or alternatively, can be produced for multiple mathematical combinations simultaneously, i.e., a multi-dimensional analysis.
In a presently preferred embodied form, the inventive apparatus comprises, in combination:
a) a polarized laser that produces a beam waist;
b) an optical chassis including multiple light detectors, each light detector positioned around and oriented to view, without obscuration, a common region of regard of the laser beam waist;
c) a sample chamber for containing a fluid sample to be analyzed;
d) means for holding the sample chamber in a prescribed orientation with respect to the laser beam waist and in the common region of regard of the light detectors;
e) means for causing the particles in the sample to circulate through the laser beam waist;
f) means for covering the light source and optical chassis to create a dark enclosure;
g) means for converting the light intensity values measured by the detectors into digital values;
h) means for continuously entering the digital values into a computer;
i) means for determining when a particle has entered the light beam at the common region of regard based on the digitized measurements;
j) means for converting the digitized values to calibrated values;
k) means for extracting Event Descriptors from the digitized and calibrated event data;
l) means for calculating Discriminant Function values from the Event Descriptors;
m) means for defining probability histograms that enable the calculation of the probability that a Discriminant Function value calculated from measured values was caused by a specific particle species;
n) means for identifying the most effective Discriminant Functions.
o) means for storing the probability histograms and Discriminant Functions in an Identification Library, one histogram for each particle species that can be identified and each Discriminant Function;
p) means for retrieving previously stored probability histograms and Discriminant Functions, one probability histogram for each particle species that can be identified with the Identification Library and each Discriminant Function;
q) means for calculating the probability for each particle species in the library for a given value of a Discriminant Function;
r) means for combining probabilities for each particle species that can be identified with the Identification Library; and
s) means for identifying the unknown particle based on a threshold.